11
30 BEST PRACTICES IN LC-MS METHOD DEVELOPMENT AND VALIDATION FOR DRIED BLOOD SPOTS Jie Zhang, Tapan K. Majumdar, Jimmy Flarakos, and Francis L.S. Tse 30.1 INTRODUCTION A major challenge for the pharmaceutical industry is finding new ways of increasing efficiency while reducing the cost of drug development. One area of opportunity in the research and development of new chemical entities is the sampling method for exposure assessments. In recent years, there has been a significant increase in the number of published reports related to the dried blood spot (DBS) sampling technique in drug development (Li and Tse, 2010; Majumdar and Howard, 2011). Some of the key features of DBS technology are the use of low blood volume potentially leading to decreased animal use, the ease of sample collection that does not require accurate blood volume measurements, and the low cost of sample storage and shipment obviating the need for refrigeration or freezing. In addition, a dry matrix is also considered less pathogenic than its corresponding liquid. As a result, DBS provides much desired ethical and possibly financial advantages over conventional blood sample handling. Significant progress has been made by the bioanalytical community to better understand the DBS technology (Ji et al., 2012; Viswanathan, 2012). Aspects related to the unique DBS procedures or parameters such as DBS assay sensi- tivity, drying process, storage and transportation at ambient temperature, dilution integrity, and incurred sample reanal- ysis (ISR) have been explored and well managed. However, some technical issues including DBS homogeneity, blood hematocrit (HCT), and extraction recovery remain to be resolved before the technique can be universally applied. The US FDA currently does not accept stand-alone DBS data as a replacement for liquid matrices in any registration studies. Although the approaches used in method development and validation of dry matrices are similar to those used for liquid biomatrices, the overall experimental protocol will need to be adapted to manage a unique set of scientific challenges posed by DBS. DBS methods must be developed and validated to meet the same acceptance criteria such as precision, selectiv- ity, sensitivity, reproducibility, and stability, which are man- dated by the health regulatory authorities for liquid matrices. In addition, many DBS specific parameters including HCT and homogeneity must be evaluated. There are extensive ongoing discussions within the bioanalytical community regarding how DBS method validation should be performed. In 2011, European Bioanalysis Forum (EBF) published its recommendations on the validation of bioanalytical methods for DBS in a white paper (Timmerman et al., 2011). This chapter provides an overview on the best practices in method development and validation for DBS as applied today. It is worth stating that although whole blood is the most commonly used matrix when referring to DBS technology, other matrices such as plasma, serum, and urine have also been evaluated in the dry form. Therefore, the general term DMS (dried matrix spot) is also used when referring to the technology. In addition, automation based, direct elution, and direct surface desorption techniques have been devel- oped in order to manage the tedious manual punching and off-line extraction steps that are potential impediments to the analytical benefits provided by DBS techniques. DMS and direct analysis of DBS are out of the scope in this chapter. Handbook of LC-MS Bioanalysis: Best Practices, Experimental Protocols, and Regulations, First Edition. Edited by Wenkui Li, Jie Zhang, and Francis L.S. Tse. C 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc. 379

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30BEST PRACTICES IN LC-MS METHOD DEVELOPMENTAND VALIDATION FOR DRIED BLOOD SPOTS

Jie Zhang, Tapan K. Majumdar, Jimmy Flarakos, and Francis L.S. Tse

30.1 INTRODUCTION

A major challenge for the pharmaceutical industry is findingnew ways of increasing efficiency while reducing the cost ofdrug development. One area of opportunity in the researchand development of new chemical entities is the samplingmethod for exposure assessments. In recent years, there hasbeen a significant increase in the number of published reportsrelated to the dried blood spot (DBS) sampling techniquein drug development (Li and Tse, 2010; Majumdar andHoward, 2011). Some of the key features of DBS technologyare the use of low blood volume potentially leading todecreased animal use, the ease of sample collection that doesnot require accurate blood volume measurements, and thelow cost of sample storage and shipment obviating the needfor refrigeration or freezing. In addition, a dry matrix is alsoconsidered less pathogenic than its corresponding liquid. Asa result, DBS provides much desired ethical and possiblyfinancial advantages over conventional blood samplehandling.

Significant progress has been made by the bioanalyticalcommunity to better understand the DBS technology (Ji et al.,2012; Viswanathan, 2012). Aspects related to the uniqueDBS procedures or parameters such as DBS assay sensi-tivity, drying process, storage and transportation at ambienttemperature, dilution integrity, and incurred sample reanal-ysis (ISR) have been explored and well managed. However,some technical issues including DBS homogeneity, bloodhematocrit (HCT), and extraction recovery remain to beresolved before the technique can be universally applied.The US FDA currently does not accept stand-alone DBS

data as a replacement for liquid matrices in any registrationstudies.

Although the approaches used in method development andvalidation of dry matrices are similar to those used for liquidbiomatrices, the overall experimental protocol will need to beadapted to manage a unique set of scientific challenges posedby DBS. DBS methods must be developed and validated tomeet the same acceptance criteria such as precision, selectiv-ity, sensitivity, reproducibility, and stability, which are man-dated by the health regulatory authorities for liquid matrices.In addition, many DBS specific parameters including HCTand homogeneity must be evaluated. There are extensiveongoing discussions within the bioanalytical communityregarding how DBS method validation should be performed.In 2011, European Bioanalysis Forum (EBF) published itsrecommendations on the validation of bioanalytical methodsfor DBS in a white paper (Timmerman et al., 2011). Thischapter provides an overview on the best practices in methoddevelopment and validation for DBS as applied today.

It is worth stating that although whole blood is the mostcommonly used matrix when referring to DBS technology,other matrices such as plasma, serum, and urine have alsobeen evaluated in the dry form. Therefore, the general termDMS (dried matrix spot) is also used when referring to thetechnology. In addition, automation based, direct elution,and direct surface desorption techniques have been devel-oped in order to manage the tedious manual punching andoff-line extraction steps that are potential impediments tothe analytical benefits provided by DBS techniques. DMSand direct analysis of DBS are out of the scope in thischapter.

Handbook of LC-MS Bioanalysis: Best Practices, Experimental Protocols, and Regulations, First Edition. Edited by Wenkui Li, Jie Zhang, and Francis L.S. Tse.C© 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.

379

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380 BEST PRACTICES IN LC-MS METHOD DEVELOPMENT AND VALIDATION FOR DBS

30.2 METHOD DEVELOPMENT

Method development is critical in establishing a solidfoundation for a validated bioassay, and it plays a key role inpromoting DBS technology. Method development for DBSbioanalysis begins with the collection of basic physiochem-ical information about the compound (analyte) such asmolecular weight, polarity, ionic character, pKa values andsolubility. If a bioanalytical method for an analyte is alreadydeveloped for a liquid matrix, one can usually modify andapply it to DBS.

A standard bioanalytical approach for working with DBStypically consists of three parts: (1) preparation of spikedblood samples (i.e., calibration standards and quality controls(QCs)) in the liquid form using fresh blood with controlledHCT at a normal level; (2) spotting blood sample onto aDBS card or paper substrate. In this step, a small volumeof blood sample between 15 and 30 μl is spotted onto apredefined type of DBS card followed by drying for about 2 hor more under controlled ambient temperature and humidityin the laboratory; (3) after drying, a disk, typically 3–8 mmin diameter, is punched out of the card, and sample extractionfrom the punched disks is performed with an organic solventin the presence of water. The volume of extraction solvent isrelatively small, ca. 100 μl. Internal standard (IS) is normallyadded to DBS samples during extraction. The extract is thenanalyzed by LC-MS/MS.

30.2.1 Preparation of Calibration Standard and QCSamples in Whole Blood

The analyte(s) is spiked into the blood matrix with an appro-priate amount of stock or working solution. To reduce thelikelihood of variation between the incurred samples, spikedcalibration standards and QC samples due to the presence ofnonendogenous organic and/or aqueous solvents, it is recom-mended to prepare a working intermediate in plasma, that is,a mixture of analyte stock solution and plasma. This is typi-cally accomplished by spiking a stock solution in plasma ata 50-fold higher concentration than the target ULOQ (upperlimit of quantification) of the assay. Calibration standardsand QC samples are then derived by serial dilutions of theintermediate with blood to achieve the target concentrationsfor the DBS samples. The amount of external components,for example, solvents added to the blood should be as littleas possible (typically < 5% of the final volume) to preventsolvent effects creating differences between spiked versusincurred samples. In addition, inappropriate dilution to bloodmatrix with external components may lead to changes in thenature of the spot formation, distribution of the compoundon the filter paper, hemolysis of blood cells prior to applyingto the filter paper, or the drying time.

Only fresh blood matrix is used for the preparation ofcalibration standards and QC samples. Fresh blood means

that it is harvested on the day of use. However, as this is notfeasible for many bioanalytical laboratories, it is also com-monly acceptable to use blood within 2 weeks of collectionand stored in a refrigerator without freezing. It is suggestedto inspect the stored blood for appropriate quality prior touse, as clotting will begin over time and can have an impacton spot size and appearance of blood on DBS substrates. Useof hemolyzed blood for preparation of calibration standardsand QC samples should be avoided as its potential impact onthe quantification of DBS is difficult to predict.

To ensure robustness and reproducibility in bioanalyticalassays, attention should be given to the integrity of spikedblood samples. Blood should be handled gently duringsample dilution. Vigorous shaking of blood may inducehemolysis and should always be avoided. An appropriateequilibration period may be critical during serial dilution inblood and prior to application of the spiked sample onto thecard matrix.

DBS results are subject to influence by the HCT value ofthe blood matrix. HCT, packed cell volume or erythrocytevolume fraction is the percentage of blood volume that isoccupied by red and white blood cells. HCT level is normallyabout 40–45% for men and women and changes with age,sex, and general health condition. A range of 28–67% HCTgenerally covers the majority of juvenile and adult humanblood samples likely to be encountered by an analyticallaboratory, except for samples from subjects with certainmedical conditions such as polycythemia and anemia (Shan-der et al., 2011). Differences in blood HCT level are reflectedin different blood viscosity values, leading to differencesin flux and diffusion properties of blood through differentsubstrates used for DBS sample collection. The distributionof blood with a high HCT through the paper substrate is lessthan that of blood with a low HCT, resulting in a smallerblood spot. Therefore, DBS samples of the same punchsize but prepared using blood samples with different HCTlevels can generate a noticeable bias in analytical results. Inaddition, different HCT levels may impact the aging of theDBS spots and also the recovery of analyte extraction fromDBS cards. It is, therefore, important to define the HCTlevel during method development and validation. The HCTlevel should be within the range anticipated in the targetpopulation of the planned study. When purchasing bloodfrom vendors for the preparation of calibration standard andQC samples, the HCT levels should be noted. Adjustmentscan be made in the bioanalytical laboratory by addingwashed and packed blood cells or plasma.

30.2.2 Selection of DBS Card

Commercially available DBS cards can be grouped accordingto the card material and chemical treatment as follows:

1. Untreated cards: Whatman DMPK-C (GE Health-care Bio-sciences, NJ, USA) and Ahlstrom 226 (ID

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METHOD DEVELOPMENT 381

Biological systems, currently part of Perkin Elmer,SC, USA).

2. Chemically treated cards: Whatman FTA DMPK Aand FTA DMPK B. The chemical(s) in the cards arethe protein denaturing agents that allow inactivation ofendogenous enzymes present in biological matrices.The treatments also prevent the growth of bacteria andother microorganisms. The difference between DMPKA and DMPK B is in the blood spot area (DMPK Acard is ∼20% smaller than DMPK B card).

3. Noncellulose cards: Agilent Bond Elute noncellulosematrix spotting cards (Agilent Technologies, Inc., CA,USA). According to the manufacturer, the Bond EluteDBS card can reduce the variability of blood spot sizecaused by varying HCT levels.

The impact of the HCT and punch location effect were dif-ferent depending on the type of DBS card. In a test using fivecompounds with different physicochemical characteristics,O’Mara et al. (2011) found that when comparing HCT effecton bias of individual compounds across card types, the What-man FTA DMPK B card type produced a notably smallernumber of acceptable biases for all compounds tested, par-ticularly when compared with the DMPK A card type. Ina plot of percentage bias against HCT level for individualcompounds on a specific card type, the slopes were foundto be dependent on not only the compound but also thecard type. This compound dependency was more obviouson untreated card Ahlstrom 226 and Whatman DMPK C.At a HCT level of 0.45, for all compounds on three of thecard types (Ahlstrom 226, DMPK A and DMPK C) tested,the concentration of compound measured in the perimeterpunches was greater than that measured in the center punch.In contrast, all compounds tested on DMPK B card werealmost homogeneously distributed across the spot. For com-pounds spotted on untreated cards (Ahlstrom 226 and What-man DMPK C), the range of bias related to heterogeneousdistribution of compound across the spot was greater thanthat compared with chemically treated card types (DMPK Aand B).

Additional criteria to be considered for DBS card selec-tion include the matrix effect, ion suppression, and recoveryof the method. Using acetaminophen and its major metabo-lites as model compounds, Li et al. (2012b) determined thematrix effect and recovery results from five types of DBScards (Ahlstrom 226, FTA Elute micro, FTA DMPK A, FTADMPK B and Agilent Bond Elute cards). After applyingblank blood on each card type followed by a drying period, a3-mm disk (in three replicates at three concentration levels)was taken from each card type for extraction. Neat solutionsof the analytes at known concentrations (same as the low,mid, and high QC samples) were spiked, in three replicates,into the blank DBS sample extracts. Subsequent LC-MS/MS

analysis showed Ahlstrom 226 card to have the best consis-tency in extraction recovery.

30.2.3 Spotting Volume, Spotting Techniques,and Punch Size

The accurate determination of analyte concentration in DBSdepends on the homogeneity of blood spreading across DBScard sampling area, spotting techniques of laboratory per-sonnel, and consistency of the punch taken from the spot.Liang et al. (2011b) reported on the impact of blood spottingvolume on the bioanalysis of dextromethorphan and dextror-phan using DMPK B card. Concentration differences up to∼19% were observed for the two analytes when the spottingvolume was changed from 10 to 50 μl. On the other hand,using the central portion of the blood spots on FTA Elutecards, Clark et al. (2010) demonstrated an even distributionof 14C-labeled compound UK-414495 in spotting volumesbetween 15 and 45 μl. These results suggest that the impactof spotting volume on the DBS assay may be compounddependent.

A small sample volume, typically 15–20 μl, is a goodstarting volume in method development. The reproducibilityof the assay is then assessed at varying volumes, typically± 50% of the target volume. Overloading of blood on DBScards could lead to heterogeneous spot formation. To provideguidance on DBS sample collection to the clinical laborato-ries, it would be helpful to determine a spot size bound-ary, especially if spotting on card is done without accuratepipetting.

30.2.4 Spot Homogeneity

Spot homogeneity refers to the distribution profile of analyteacross the DBS. Since it can have a significant impact onassay precision, accuracy, and reproducibility, spot homo-geneity should be evaluated during method development.The factors that govern spot homogeneity include the typeof filter paper (card) and the physiological parameters of theblood sample such as HCT and drying conditions. In addition,chromatographic effect during spotting can change the wayan analyte moves in the filter paper, with paper acting as thestationary phase and the liquid matrix as the mobile phase.Depending on the structure of a given analyte, a chromato-graphic effect can drive the analyte to the edge of the spot, orconcentrate it in the center of the spot. Using an autoradio-gram technique, Ren et al. (2010) found uneven distributionpatterns for compounds across the DBS, particularly for rela-tively large spot volumes (100 μl). This effect can cause biasif the same portion is not reproducibly removed betweenspots or when two samples from different locations on thesame spot were removed for analysis. Therefore, one shouldaim to take larger punch samples from the same location ofa spot whenever possible.

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382 BEST PRACTICES IN LC-MS METHOD DEVELOPMENT AND VALIDATION FOR DBS

The experimental design for determining spot homogene-ity is to compare the concentrations of the same analyte in thepunches from different locations of the card containing thesame QC samples at several concentration levels (typicallylow, mid, and high QC levels). Alternatively, in cases wherea card size is not sufficiently large for multiple punches, con-centration determinations of the same analyte can be madeby comparing punches of the same QC samples from dif-ferent cards. A good reproducibility (<15 %CV) derivedfrom analysis of the QC samples along with a set of calibra-tion standards would demonstrate the comparability of eachpunch and card.

30.2.5 DBS Sample Drying, Storage, andTransportation

After spotting on a DBS card, the spotted card is placed ona drying rack at ambient temperature and normal humidity.The drying time depends on the type of card, blood volumeapplied, and humidity. Based on a thorough evaluation onDBS storage conditions with the Whatman 903, FTA andFTA Elute cards using weight and appearance of DBS as ref-erences, Denniff and Spooner (2010) suggested that bloodspots should be left to dry for at least 90 min under ambi-ent laboratory conditions prior to further handling or anal-ysis. It was noticed that the three types of cards examinedbehaved differently when exposed to conditions of high rel-ative humidity and temperature. The exposure of FTA andWhatman 903 substrates to high relative humidity and tem-perature did not adversely affect the blood spot, whereas thespots on the FTA Elute expanded and diffused through thesubstrate over 24 h under the same conditions, suggesting thatthe integrity of the DBS samples has been compromised.

Storage of biological samples on DBS is more convenientthan those in liquid matrices. Generally, DBS card storageat room temperature would suffice, although a lower temper-ature may be necessary for extending stability coverage forsome analytes. Typical degradation reactions (reduction, oxi-dation or hydrolysis) are expected to be 10 times slower whenthe temperature is decreased from 22◦C to 0◦C (Chen andHsieh, 2005). Nonetheless, many analytes unstable in liquid

formats upon storage have shown enhanced stability in DBS(Li and Tse, 2010). Water in liquid matrices plays a criti-cal role in enzymatic and chemical hydrolysis reactions thatcleave drug molecules (Alfazil and Anderson, 2008). Watermay also induce bacterial growth on DBS substrates that canalter extraction efficiency during analysis. DBS cards shouldbe packed in zip-closure bags with desiccant packages andhumidity indicator cards to ensure protection from moisture.

With generally established stability at ambient and highertemperatures, DBS samples can be shipped via conventionalcarriers without dry ice or refrigeration. The savings in ship-ping costs could be significant particularly during late-phaseclinical trials that can generate a large number of samples.

30.2.6 Internal Standard

A structurally related compound, a structurally similar com-pound or a stable isotope labeled compound at a specificconcentration is added to all samples in an analytical run asan IS to correct for any variability during sample preparationand analysis using mass spectrometric detection. When ana-lyzing liquid samples such as plasma or serum, IS is simplyadded by spiking into the sample prior to extraction. In con-trast, the addition of an IS in the analysis of DBS samples issomewhat complicated.

There are generally four options for introducing an IS inDBS method development as shown in Figure 30.1:

a. Introduction of IS via an extraction solvent. This sim-ple technique is widely used and works reproduciblyby compensating for matrix effects and differences inextraction efficiency, losses due to sample handling andvariations in instrument sensitivity. A potential flawof this method is that the IS may not be fully incor-porated into the matrix components and sample card,thus unable to compensate for fluctuations in extractionfrom the DBS filter punch. In addition, this approachmay not reveal changes in extraction recovery due tostorage.

Wholeblood

BlankDBScard

SampleDBScard

DBSdisk

Extractionsolvent

++

C B AD

PunchingSpotting

FIGURE 30.1 Four points where an internal standard can be added to DBS samples in LC-MS/MSanalysis: (a) Introduction of IS via an extraction solvent; (b) DBS card pretreated with IS; (c) ISadded to blood before spotting onto paper; (d) IS applied to DBS matrix prior to extraction.

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METHOD DEVELOPMENT 383

b. DBS card pretreated with IS. By incorporating blankDBS cards with IS prior to applying the blood sam-ples, the IS is integrated and extracted with the analyte.However, pretreating the DBS cards prior to samplecollection is tedious and costly. The potential impacton IS migration or distribution on the DBS card duringmatrix spotting also needs to be clearly characterized(Meesters et al., 2011).

c. IS added to blood before spotting onto paper. Theadvantage of this approach is that the IS can be fullyassociated with blood components together with theanalyte. However, this procedure may be too com-plicated to implement at most clinical sites. It willalso require extra safety measures when sampling frompatients with infectious diseases.

d. IS applied to DBS matrix prior to extraction. This is anovel technique pioneered by Abu-Rabie et al. (2011)using the TouchSpray technology to apply IS to DBSsamples prior to analysis. The IS must be given suffi-cient time prior to extraction to bind with matrix com-ponents and paper substrate and must not adverselyaffect the distribution of the analyte. There was no sig-nificant difference in accuracy and precision obtainedfrom this procedure compared with procedures A andC. This procedure can be easily configured to a fullyautomated method for DBS sample analysis.

Different procedures used to introduce the IS can result inmarkedly different recoveries. Meesters et al. (2011) recentlyreported that the relative recovery of a model compound nevi-rapine ranged between 11.4% and 108%, highlighting theneed for careful evaluation of IS procedures during methoddevelopment and validation in order to ensure assay integrity.On the other hand, the procedure for IS application shouldnot be so complicated as to negate the advantages of DBSsampling.

30.2.7 Extraction Solvent, Procedure, and Recovery

The objectives of sample preparation for any bioanalyticalassay are to maximally remove the matrix background andinterferences, efficiently recover the analytes of interest andISs, while maintaining an adequate sensitivity of the assaymethod. Changing the sample format from liquid to solidphase is accompanied with a unique set of challenges in thesample extraction process.

A common extraction procedure for DBS samples is topunch one or more DBS disks from the DBS card into tubes or96-well plates followed by adding a finite amount of extrac-tion solvent containing the ISs. The analyte of interest isthen extracted with gentle shaking or vortex mixing. Son-ication may be necessary to enhance extraction efficiency.After centrifugation, the resulting extracts are transferred

manually or by an automated liquid handler to fresh tubesor 96-well plates for LC-MS/MS analysis. The extractionsolutions should be able to interrupt the binding of analyte tothe matrix proteins and the paper material. Several organicsolvents or their mixtures with water may be consideredinitially, for example, methanol, methanol:water, or acetoni-trile:water, at various ratios. The water in the extraction solu-tion may be helpful for effectively eluting the analytes off theDBS card. Pure acetonitrile may not dissolve the dried bloodcrust completely, resulting in poor extraction of the analyteand low elution efficiency (Liu et al., 2010). Depending onthe structure of the analyte, a pH modifier or buffer may beadded to the extraction solvent to improve its efficiency. Sev-eral iterations may be needed to reach the optimal organicsolvent:water ratio in the extraction solution that will pro-vide maximum extraction efficiency and minimum matrixeffect.

An alternative sample preparation approach is to add theorganic and aqueous portions of the extraction solution intwo steps. First, an aqueous solution is added to DBS disksto dissolve the dried blood, and then acetonitrile or methanolto precipitate the proteins that were eluted from the DBScards. This approach is similar to the protein precipitationmethod commonly used for liquid sample analysis. If a water-immiscible organic solvent such as methyl tert-butyl ether orethyl acetate is added to the aqueous solution, the procedurewould be comparable to the liquid–liquid extraction (LLE)approach for liquid samples. Of the above, LLE appears to bethe most effective extraction method in removing the matrixbackground introduced by DBS cards (Liu et al., 2010).

Determination of extraction recovery of analytes fromblood samples spotted on a DBS card is a challenging task.Understanding the nature of analyte interactions with thesubstrate is prerequisite to establishing the procedure foroptimal extraction. Other factors that may impact analyterecovery include HCT level, age of paper substrate, tem-perature, and humidity. In most DBS assays, the IS is addedduring sample extraction, thus the elution of the analyte fromthe DBS card (solid phase) to the extraction solution (liquidphase) is not monitored by the IS. It is, therefore, importantto elucidate the efficiency of the extraction process duringmethod development. If available, a radiolabeled drug sub-stance may be used to monitor analyte extraction from theDBS card.

The current FDA guidance for liquid sample assays statesthat “the extent of recovery of an analyte and of the inter-nal standard should be consistent, precise and reproducible.Recovery experiments should be performed by comparingthe analytical results for extracted samples at three concentra-tions (low, medium and high) with unextracted standards thatrepresent 100% recovery” (FDA, 2001). EBF recommendedevaluating and documenting recovery more thoroughly forDBS assays than liquid sample assays. In particular, EBFbelieves that it is important to evaluate the potential impact

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384 BEST PRACTICES IN LC-MS METHOD DEVELOPMENT AND VALIDATION FOR DBS

of card storage on extraction recovery from the punched disksas part of the test for long-term stability (EBF, 2010).

30.2.8 Matrix Effects

The evaluation of matrix effect for DBS assays is similar tothat used in liquid assays. Typically, a punch from a DBScard spotted with blank matrix is made followed by extrac-tion. The analyte is then spiked into the extract at a specifiedconcentration. The extract is analyzed along with a neat solu-tion of the analyte to determine the matrix effect.

Some blood components bound to DBS paper may not bedissolved in aqueous or organic solvents, thus the extractsof DBS may be cleaner (i.e. less matrix effect) than thatderived from liquid samples. However, DBS sample extractsmay contain certain endogenous materials such as leachablechemicals that may cause high background noises or interfer-ences during LC-MS analysis. Furthermore, different typesof DBS cards may introduce different interferences into thesample extract. It is well known that treated card materi-als and/or the chemicals used to pretreat of DBS cards maycontribute to ion suppression of the analyte causing matrixeffects (Clark and Haynes, 2011)

30.2.9 Assay Sensitivity

Compared with liquid sample analysis, the sensitivity of DBSassay is likely to be lower due to the relatively small samplepunch size. One can try to enhance the signal intensity ofanalyte in DBS samples by extracting from multiple punchesor larger punched disks, and by optimizing the extractionsolvent. Recent advancements in DBS technology includethe use of microbore column and microflow in LC–MS/MSthat have shown promising success in increasing DBS assaysensitivity (Rahavendran et al., 2012; Rainville, 2011).

30.2.10 On-Card Stabilization for UnstableCompounds

Different approaches to ensure on-card stabilization shouldbe evaluated early in method development for analytessuspected to be unstable. Stabilizing unstable compoundsis a major challenge in bioanalytical method developmentfor liquid based samples, and a variety of stabilizationtechniques have been used in an attempt to protect thecompound in blood, plasma, and serum after collection (Liet al., 2011). Adding a chemical reagent to blood samplesimmediately after collection can stop the degradation orstructural changes of a drug, an example being the useof organic acids to stabilize esters and lactones. Samplescontaining light sensitive compounds should be handledand stored in dark chambers, and those with heat-sensitivecompounds stored in deep freezers. The same principlesfor handling liquid matrices also apply to DBS samples.However, DBS has an advantage over liquid-based matrices;

the water in the latter plays a key role in enzymatic andchemical hydrolysis reactions that can alter the molecularstructure of a drug (Alfazil and Anderson, 2008). One studyshowed that DBS sampling stabilized two unstable prodrugsin rat whole blood for at least 21 days at room temperaturewithout the addition of esterase inhibitors (D’Arienzo et al.,2010). Interestingly, Whatman DMPK A and B cards,despite having been treated with reagents that lyse cells uponcontact, showed no advantage in terms of added stabilityfor the investigated compounds compared to the untreatedWhatman 903 Protein Saver cards. This suggests that theon-card stabilizing effect could be compound dependent.Sometimes, it may be necessary to modify the commerciallyavailable DBS cards to meet the stabilization needs ofotherwise unstable compounds. For example, Liu et al.(2011b) pretreated DBS cards with citric acid to lower thepH of the spotted blood sample containing an unstable drugcandidate KAI-9803 which consisted of two peptides linkedby a disulfide bond. The result was an improved compoundstability in DBS to at least 48 days at room temperature.

In another study, a photosensitive compound omeprazoleexhibited increased stability when spotted and stored on DBSpapers (Bowen et al., 2010). This compound degraded 40-90% in water, plasma or whole blood, whereas photodegra-dation was negligible in DBS.

30.3 METHOD VALIDATION

Method validation is conducted to confirm that an analyticalprocedure established during method development is suit-able for its intended use. A well-developed method shouldbe easily validated. Failure to meet the preset criteria duringmethod validation requires a thorough investigation to under-stand the root cause of the problem. The knowledge gainedfrom working with liquid matrix samples also applies to DBS.

At the current stage of DBS technology development,method validation is usually performed based on a fit-for-purpose concept since no official guidance is available. Sim-ilar to the comprehensive evaluation for a liquid matrix,DBS assay validation includes precision, accuracy, selec-tivity, specificity, extraction recovery, matrix effects, dilu-tion, incurred sample reanalysis, and stabilities includingwhole-blood collection stability. The characterization ofthese parameters is made according to the existing guide-lines on bioanalytical method validation (EMA, 2011; FDA,2001). Additional validation for DBS specific parameterssuch as spot drying stability, HCT impact, inter- and intrac-ard variability must be performed.

30.3.1 Selectivity, Sensitivity, and Linearity

The selectivity of a DBS method is usually assessed byanalyzing DBS samples prepared from fresh blank blood

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METHOD VALIDATION 385

collected from at least six individual human subjects or atleast two individual animals. The lower limit of quantifica-tion (LLOQ) must exhibit an accuracy of within ± 20% biasand precision of CV ≤20%. At least six nonzero calibrationstandards are analyzed in duplicates (typically one in thebeginning and the other at the end of the assay sequence)in three separate validation runs. The analyte/IS peak arearatio against nominal analyte concentration is employedfor calibration regression with an appropriate weightingfactor.

30.3.2 Inter- and Intraday Accuracy and Precision

The inter- and intraday accuracy and precision of the DBSmethod are demonstrated from the analysis of 6 replicates ofQCs at a minimum of four concentrations levels includingone at the lowest concentration of the calibration standards oneach of the three validation runs. The accuracy is expressedas the difference of the measured analyte concentrations fromthe nominal values (bias %) and the precision as the coeffi-cient of variation (CV %). A bias of within ± 15% and CVof ≤15% is required at all concentration levels except theLLOQ, for which a ± 20% bias and ≤20% CV are consid-ered acceptable.

30.3.3 HCT and Its Effects on the Assay

As stated in the EBF recommendation on DBS (Timmer-man et al. 2011), “hematocrit is currently identified as thesingle most important parameter influencing the spread ofblood on DBS cards, which could impact the validity of theresults generated by DBS methods, affecting the spot forma-tion, spot size, drying time, homogeneity and, ultimately, therobustness and reproducibility of the assays.”

The EBF recommends that “for bioanalytical method val-idation, the impact of variations of HCT on the spot sizeand homogeneity should be understood and their impact onassay performance documented during validation. For that,clinically relevant variations of HCT (e.g., from 30–35% to55–60%) should be evaluated during validation. Patients withphysiological conditions or under medical treatment affect-ing the HCT beyond normal values (e.g., renal impairmentand oncology patients) may require additional validation asthey occur, such as including calibration standards and qual-ity control samples prepared using matrix beyond normalHCT values.” Viswanathan (2012) suggested that subsequentto acceptable homogeneity determinations, efforts need tobe made to avoid or minimize the assay bias with differ-ent ranges of HCT values between calibration standards andquality control samples. An optimal range in this regard maybe established during validation such that the range in thestudy samples can be accommodated. Thus, the investiga-tion and the evaluation of the above parameters on the overall

impact of DBS will be considered as the central part of thevalidation.

To determine the influence of HCT on the assay perfor-mance, fresh blood with adjusted HCT values of 30%, 40%,50%, and 60% may be obtained either from a commercialsource or prepared in the bioanalytical laboratory. These freshblood samples are each used to prepare DBS QCs at concen-trations of low and high concentration levels (n = 6 QCs ateach level). The QC samples are analyzed in the validationruns along with the calibration standards and QC samplesprepared using fresh blood with a HCT value of ∼ 35%. Adifference beyond ± 15% of the nominal analyte concentra-tions in the QC samples would suggest a significant HCTeffect. It is worth noting that the HCT in some disease statesmay be out of the range (30–60%) tested.

A significant HCT effect can be managed by the following:

1. Correcting concentration results using each subject’sHCT value: To do this, correction factors must beestablished for each HCT value relative to the HCTof the blood used to prepare the standard curve dur-ing method validation. The HCT value for each studysubject needs to be determined and this could add asignificant burden to the clinical program.

2. Analyzing the entire spot: This approach yields moreconsistent DBS concentrations regardless of HCT lev-els by eliminating the variations due to spreading andnonhomogeneity. The challenge to this approach isthat it requires the accurate spotting of a defined bloodvolume. Li et al. (2012a) described a novel procedurecalled perforated dried blood spot (PDBS), in which anaccurate amount of blood (5–10 μl) was added, usingeither a Micro Safe pipette or a Drummond incremen-tal pipette on the PDBS disks prepared from regu-lar filter paper (6.35 mm in diameter and a 0.83 mmin thickness). PDBS samples are simply pushed bysingle-use pipette tips into 96-well plates for analy-sis. This technique provides a promising solution tothe adverse impact of HCT on DBS analysis by using100% of the sample.

30.3.4 Impact of Blood Volume and Spot Size on theAccuracy of Determination

During DBS method validation, the accuracy of determina-tion for an analyte from DBS samples with variable samplingspot sizes is examined by spotting increasing volumes of DBSQC samples (typically 10, 20, and 40 μl) at three concentra-tions (low, median, and high) onto the cards. After drying,three replicates of 3-mm disks are taken from the center ofeach DBS QC sample and analyzed along with calibrationstandards. A bias within ± 15% of the nominal values wouldsuggest no apparent difference for the DBS samples made

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386 BEST PRACTICES IN LC-MS METHOD DEVELOPMENT AND VALIDATION FOR DBS

with different blood volumes. This would indicate an evendistribution of blood on the card so that precise sampling oncard (over the range of blood volume evaluated) may not benecessary.

30.3.5 Impact of Homogeneity on the Accuracyof Determination

The possible effect on blood diffusion by interactions ofblood and/or the analyte with the DBS card materials isassessed by punching the DBS disks from the center and edgearea of DBS QC samples at low, mid, and high concentra-tions, followed by analysis along with calibration standards.The measured analyte concentrations from both the centerand edge disks of the above QCs were compared againsteach other and also with the nominal values. Bias within± 15% of the nominal concentration and within ± 15% ofeach other would suggest no apparent chromatographic effect(i.e., no impact on spreadability).

30.3.6 Temperature Impact

DBS QC samples at low, mid, and high concentrations areanalyzed each in three replicates following storage at roomtemperature and 2–8◦C. The measured analyte concentra-tions are compared with the nominal values. The calculatedbias (%) from the stability QCs should be within ± 15% ofthe nominal values. To mimic the possible situation where theDBS samples are collected and/or transported at a high tem-perature, a set of DBS QCs may be stored at a temperature upto 70oC for several hours (e.g., 4 h) followed by analysis withcalibration standards and regular QCs. Bias within ± 15%of the nominal values would suggest the analyte is stable inthe DBS sample at elevated temperatures.

30.3.7 Dilution Integrity

Dilution integrity needs to be evaluated during method vali-dation in order to ascertain that a method is suitable for thebioanalysis of DBS samples containing analyte concentra-tions higher than the ULOQ. While the principles of dilutionin the traditional liquid sample analysis apply to DBS, thedilution procedures for DBS samples are more complicateddue to its solid format. Three common approaches to DBSsample dilution are as follows:

1. Dilution with blank DBS extract: In this procedure,the extract of a DBS sample from the punched diskis diluted with one or multiple extracts of blank DBSsamples (the number of blank extracts used = the dilu-tion factor − 1). An IS can be added either to theextraction solvent prior to the dilution or to the dilutedextract. This method requires a great deal of blank

matrix and processing additional DBS cards in eachanalytical run. It works well when a relatively smalldilution factor is needed for a few samples, but canbe costly or impractical for samples requiring largedilution factors.

2. IS-tracked dilution: In this approach introduced byLiu et al. (2011a), a dilution factor-adjusted IS work-ing solution is added to the sample requiring dilutionprior to sample extraction. Subsequently, the processedsample is approximately diluted to the assay linearresponse range for LC-MS/MS analysis. As shownin Figure 30.2, the dilution factor-adjusted IS work-ing solution is an IS working solution at a concentra-tion that is 10 times (i.e. the intended dilution factor)higher than that used for regular samples. The advan-tage of this approach is that the dilution is tracked bythe IS and is no longer a volume-critical step oncethe concentrated IS working solution is added to asample. The main disadvantage of this method is thatstandards, QCs, and study samples not requiring dilu-tion are treated differently from diluted study samples.Therefore, it is important to closely monitor the per-formance of dilution QC samples in the analytical run.

3. Subpunch dilution: In this procedure pioneered byAlturas Analytics (Christianson et al., 2011), there arethree critical steps in punching the DBS sample cardand DBS blank card. (i) A fixed diameter subpunchis collected from a DBS sample to be diluted. (ii) Anidentical size of subpunch in a blank DBS card is takenand discarded. (iii) A regular-sized punch is taken fromthe blank DBS card. The punch from the blank cardis then extracted together with the subpunch from thesample. A dilution factor is derived using mathemati-cal calculations based on the sizes of the regular punchand subpunch.

30.3.8 Intercard Variability

DBS cards are manufactured under well-defined procedures,and the physical characteristics and chemical additives areexpected to be identical among different lots of cards. Thereis consensus in the bioanalytical community that one sin-gle DBS is considered one sample. Any additional spotoriginating from the same liquid sample and spotted eitheron the same card or on a different card from the sametype/manufacturer may be treated as an identical replicatesample, provided the handling and storage conditions areidentical. EBF recommended in their white paper that whenusing cards from the same type/manufacturer, intercard vari-ability does not need to be investigated as a discrete methodvalidation parameter. However, it is a good practice to spreadcalibration standards and QC samples over multiple cardsduring validation, in order to identify or preclude intercard

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METHOD VALIDATION 387

Blankmatrix

(a) Conventional Dilution

(b) IS-Tracked dilution

Blankmatrix

Dilutedsample(1/10)

Sampleprocessing

Sampleprocessing

Processeddiluted

sample (1/10)

Processeddiluted

sample (1/10)

Processedblank sample

Processedsample

sampleProcessing

50 μl–20 μl

–20 μl

–180 μl

–180 μl

50 μl

50 μl

Dry down/reconstitute

Dry down/reconstitute

LC-MS

LC-MS

Sample tobe diluted

Sample tobe diluted

IS (10 ×)

IS

FIGURE 30.2 Two sample dilution processes for DBS sample analysis using LC/MS/MS: (a)Conventional sample dilution; (b) IS-tracked dilution. Shown here is an example for a dilution factorof 10 (reproduced from Liu et al., 2011a, with permission from Wiley).

variability issues when a validation run fails to meet prede-fined acceptance criteria (Timmerman et al., 2011).

Changing a DBS card type/manufacturer requires a par-tial validation of the bioanalytical method. The validationparameters recommended by EBF are linearity and sampledilution, accuracy and precision, extraction recovery, matrixeffects, drying conditions (i.e., drying time and temperature),and oncard storage stability.

30.3.9 Stability Determination

The principles of stability assessment for liquid samples areapplicable to DBS, which means that “evaluation of stabilityshould be carried out to ensure that every step taken duringsample preparation and sample analysis, as well as the storageconditions used do not affect the concentration of the analyte”(EMA, 2011). DBS stability evaluation is generally focusedon the following three areas in the validation:

1. Whole blood stability during collection and handlingof blood samples prior to spotting on DBS cards

2. Stability on DBS cards during and after spotting: Sta-bility at room temperature, under frozen conditionsover a certain period of time and during shipping.

Evaluation of freeze–thaw stability is not necessary,as it is not relevant for DBS samples.

3. Stability during sample preparation for LC-MSanalysis.

The stability of the analyte during blood collection andfurther handling prior to spotting on DBS cards must be estab-lished to ensure that the analytical method will yield concen-tration results that reflect the concentrations of the analyte inthe subject at the moment of sampling. The blood used forthe validation experiments should be fresh (within two weeksof collection), and the experiments should be performed atbody temperature (37◦C) to reflect the real-life situation. Theanalytical procedures for determining whole blood stabilityin liquid format can be used for DBS. Similarly, the stabil-ity of the analyte in stock and working solutions must beestablished in the method validation.

The analyte stability on DBS cards after spotting isassessed according to the intended storage conditions andfollowing various storage durations by comparing the mea-sured analyte concentrations in QC samples (at least threereplicates each of low and high QC) with the nominal val-ues. The bias should be within ± 15% of the nominal val-ues. Analyte in a matrix can be caused by hydrolysis ofthe intact conjugated molecules or other unstable conjugated

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388 BEST PRACTICES IN LC-MS METHOD DEVELOPMENT AND VALIDATION FOR DBS

molecules (e.g., acylglucuronide, O-sulfate/glucuronide, andN-oxide/glucuronide), or interaction of the analyte withendogenous components. Therefore, DBS prepared fromincurred samples should be used for stability evaluationwhenever possible. Accordingly, the analyte concentrationsmeasured from the study samples (minimum 20 samplesin single determinations) initially and after various storageperiods are compared. The difference between the repeatedmeasurement and the mean of two measurements (first andrepeat) should be within ± 20% for at least two-thirds of theselected samples.

As mentioned earlier, stability evaluations need to mimicthe conditions of sample storage. A unique characteristic ofDBS samples is that they are likely to be exposed to uncon-trolled conditions such as extreme temperatures and humidityduring sampling, sample shipment and storage, for example,conducting a clinical study in hot climates and at sites wherefreezers are not readily available. The stability profile of theanalyte under these conditions needs to be evaluated duringvalidation. Li et al. (2012b) described a validation protocolfor evaluating the possible impact of humidity and high tem-perature on acetaminophen and its major metabolites in DBSsamples. A set of DBS low and high QC samples, after dry-ing at ambient temperature in an open laboratory (humidity∼40%), was placed in sealed containers with inside humiditymaintained at ∼80% or ∼0%. Another set of the QCs wasplaced in a Shimadzu HPLC column oven with temperatureset to ∼60◦C. At 5 and 24 h, three replicates of 3-mm diskswere taken from the center of these QCs and analyzed alongwith the calibration standards and regular QCs that wereexposed to regular humidity (∼40%) at ambient temperature(∼22◦C). The measured analyte concentrations were com-pared with the nominal values.

For compounds with previously identified stability issuesin a liquid matrix, appropriate caution must be taken whenevaluating the analyte stability in DBS cards. For new com-pounds without prior stability data, studying the compoundstructure may help to predict potential stability issues (Liet al., 2011).

If lower-than-expected analyte concentrations areobserved in DBS samples, it is important to discern whether itis due to poor extraction recovery or actual compound degra-dation. An interesting approach to evaluating drug stabilityduring drying on DBS cards has been reported recently (Liuet al., 2011b).

30.3.10 Carryover

Carryover is evaluated by injecting two extracted blank DBSsamples sequentially following the injection of a sample con-taining the analyte at ULOQ. In the first blank matrix injec-tion, the response at the retention time region of the analyte orIS should be less than 20% of the mean response of the LLOQsamples for the analyte and less than 5% of the mean response

for the IS from the same assay sequence. The response in thesecond blank injection serves to provide clues that may beneeded for troubleshooting.

30.4 CONCLUSIONS

DBS is being explored as an important sampling tool inbioanalytics due to its many potential benefits. In order togain widespread acceptance for use in pharmaceutical devel-opment, however, DBS has yet to improve its reliability indelivering accurate and reproducible results over time. Con-tinued efforts are needed to overcome technological hur-dles especially in minimizing hematocrit effects, ensuringspot homogeneity, enhancing extraction recoveries of ana-lytes from DBS substrates and maximizing the stability ofunstable compounds.

Despite its perceived advantages as a sample collectionmethod, DBS sample processing in the bioanalytical labora-tory today tends to be more labor intensive compared to con-ventional, liquid matrices. No doubt there is room for processimprovements that, together with advances in automation, areexpected to make DBS bioanalysis the recognized method ofchoice in the foreseeable future.

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